Tutorial: RAREMETAL

Useful Wiki Pages

There are a few pages in this Wiki that may be useful to rareMETAL users. Here are links to a few:

• The rareMETAL Home Page

• The rareMETAL Command Reference

• The rareMETAL Documentation

• The rareMETAL FAQ

Introduction

In this tutorial, we will use RareMetalWorker and RareMetal to perform a simple rare variant meta analysis. rareMetalWorker is a tool that generates summary statistics that can be shared to enable meta-analysis of gene-level association tests. rareMETAL uses the files generated by RareMetalWorker as input and perform both single variant and gene-level association meta-analysis.

STEP 1: Install Software and Download Example Data Sets

• If RAREMETAL and RAREMETALWORKER have not been installed on your local computer yet, that is the first step! Installation instructions for RAREMETAL and RAREMETALWORKER are

available (RAREMETAL and RAREMETALWORKER).

• Then please download the tutorial package, including example data sets and results.

• • To unpack the example dataset, you can use the following two Unix commands:

 tar xvzf raremetal_tutorial.tar.gz 
 cd raremetal_tutorial

STEP 2: Analyze individual samples using RAREMETALWORKER

• The first example has 743 individuals coded as unrelated according to PED file (each person belongs to an individual family).
• there are ~1000 markers included in the VCF file.
• To analyze this sample accounting for sample relatedness, an empirical kinship should be calculated.
• Go to $yourPath/bin/ and execute the following command:

 cd raremetalworker/inputfiles

 $yourpathforRAREMETALWORKER/bin/raremetalworker  --ped example1.ped --dat example1.dat --vcf example1.vcf.gz
                                --traitName QT1 --inverseNormal --makeResiduals --kinSave --kinGeno --prefix ../yourprefix.example1

• The command above allows you to estimate relatedness according to common variants genotypes with good variant calling quality, adjust for covariates, quantile normalize the residuals before further association analysis of trait QT1. The following output are generated:

raremetalworker/yourprefix.example1.QT1.singlevar.score.txt (## summary statistics, single variant association results, QC statistics, Genomic control, etc.)
raremetalworker/yourprefix.example1.QT1.singlevar.cov.txt (## covariance matrices of score statistics)
raremetalworker/yourprefix.example1.plots.pdf (## QQ plots and manhattan plots)
raremetalworker/yourprefix.example1.Empirical.Kinship.gz (##contains empirical kinship of all individuals who have at least one site genotyped, with id on top row.)

• Following the same strategy, example2 can also be analyzed using a similar command:

  $yourpathforRAREMETALWORKER/bin/raremetalworker  --ped example2.ped --dat example2.dat --vcf example2.vcf.gz
                                --traitName QT1 --inverseNormal --makeResiduals --kinSave --kinGeno --prefix yourprefix.example2

STEP 3: Run RAREMETAL to do Meta-Analysis

• In this step, we run RAREMETAL to meta-analyze two studies without using any of raw data.
• First, we perform single variant and four type of gene-level meta-analysis all at once, using the following command line:

 cd raremetal
 $yourRAREMTALpath/bin/raremetal --studyName studyname.list --groupFile group.file --SKAT --burden --MB --VT 
                                 --longOutput --tabulateHits --hitsCutoff 1e-05 --hwe 1.0e-05 --callRate 0.95 --prefix yourprefix.QT1

• The command above allows you to gene
• A list of studies to be included is an essential piece of information for RareMETAL to run.
• First, modify the example.studyname file to make the output files of RareMetaWorker reachable by RareMETAL.

 cd $yourPath/raremetal_tutorial/inputfiles 

• Open example.studyName and modify them into the following:

 $yourLocalPath/rmw_tutorial/outputfiles/example1.LDL
 $yourLocalPath/rmw_tutorial/outputfiles/example2.LDL

• If gene-level meta analysis is expected, then annotation information or groups of variants are necessary. RareMETAL can take group file to get this piece of information. * An example group file is in the following:

 $yourLocaPath/raremetal_tutorial/inputfiles/nonsyn.stop.splice.groupfile

• RareMETAL also takes annotated VCF as input to parse variant grouping information. Please refer to software documentation for details grouping from annotated VCF
• RareMETAL allows filtering single variants to be included in meta analysis according to their QC information summarized by raremetalworker, including HWE p-value and genotype call rate.

• Finally, to meta-analyze the above two samples using summary statistics, the following command will generate results from single variant meta analysis, gene-level meta analysis using SKAT, Madsen-Browning burden test, simple burden test, Variable Threshold Burden tests.

 $yourPath/bin/raremetal --studyName --$yourLocaPath/raremetal_tutorial/inputfiles/example.studyname 
   --groupFile $yourLocaPath/raremetal_tutorial/inputfiles/nonsyn.stop.splice.groupfile --SKAT --VT --burden --MB --maf 0.05 --hwe 1.0e-05 --callRate 0.95 
   --prefix $yourLocaPath/raremetal_tutorial/results/

• To generate a lengthy results and report hits, the following command should be used:

 $yourPath/bin/raremetal --studyName --$yourLocaPath/raremetal_tutorial/inputfiles/example.studyname 
   --groupFile $yourLocaPath/raremetal_tutorial/inputfiles/nonsyn.stop.splice.groupfile --SKAT --VT --burden --MB --maf 0.05 --hwe 1.0e-05 --callRate 0.95 
   --longOutput --tabulateHits --hitsCutoff 1.0e-05 --prefix $yourLocaPath/raremetal_tutorial/results/

• Please refer to the documentation for detailed description of output format. RareMETAL Results

• RareMETAL also allows users to output an VCF file of the super set of all variants and use their favorite annotation tool to annotate it and then come back to RareMETAL for the gene-level meta analysis. --writeVCF is the option to use. Please refer to Write VCF and Annotated outside RareMETAL